Abstract Multiple sclerosis (MS) is an immune-mediated demyelinating disease of the central nervous system (CNS) which can result in severe neurological deficits. The cause of the disease is unknown, the immune and neurodegenerative mechanisms underlying the pathophysiology of the disease are poorly understood, and current therapies are only partially effective at slowing disease progression. There is a tremendous need to investigate novel mechanisms that may be contributing to disease susceptibility and the pathophysiology of this disease. To this end, my laboratory has performed a large miRNA profiling study on nave and effector/memory CD4 T cell in untreated MS patients to determine if miRNA may contribute to disease susceptibility or progression. MiRNA negatively regulate gene expression via the RNA interference pathway, and thus play a key role modulating the level of specific proteins in cells. We have identified at least two pathways that are altered in MS patients that may contribute to their susceptibility to develop MS. First, miRNAs targeting components of the Th2 cell differentiation pathway were elevated in MS patients T cells, skewing differentiation into pro-inflammatory Th1 cells. Second, miRNAs targeting the TGF? signaling pathway limited the differentiation of regulatory T cells. Thus, the defects observed in MS patients CD4 T cells may be at least partially mediated by miRNA dysregulation. In this proposal, we will address the following questions. Aim 1: Are MS patients? CD4 T cells defective in their ability to differentiate into Tregs in a miRNA-dependent manner? Preliminary data indicates that nave CD4 T cells from MS patients fail to efficiently differentiate into Tregs. Using miRNA inhibitors, we will determine if this failure to differentiate into Tregs is dependent on specific miRNAs over-expressed in MS patients and whether Treg differentiation can be normalized MS patients? nave CD4 T cells. Aim 2: How does over-expression of MS-associated miRNAs affect the development and progression of CNS autoimmunity in a mouse model? Using EAE, the role of miRNAs that target CD4 T cell differentiation into effector and regulatory T cells will be analyzed in vivo to complement the human in vitro experiments. Aim 3: Can expression level of miR-128, which targets both effector and regulatory CD4+ T cells, modulate the risk of CNS autoimmunity? We found that miR-128 targets proteins in the Th2 and TGF? signaling pathways, promoting the differentiation of Th1 cells and preventing the development of Tregs. Using CD4-specific miR-128-/- mice and mice overexpressing miR-128 in CD4 T cells, we will determine if loss of miR-128 in CD4 T cells minimizes susceptibility to CNS autoimmunity, normalizes CD4 T cell differentiation into effector and regulatory cells, and thus, is a potential therapeutic target to correct both effector and regulatory T cell defects in MS. In contrast, we will use mice overexpressing miR-128 in CD4 T cells to determine if miR-128 is sufficient to skew CD4 T cell differentiation and enhance the risk of developing CNS autoimmunity. This study will test the hypothesis that miRNA dysregulation in nave CD4+ T cells is an underlying risk factor in CNS autoimmunity that can be therapeutically targeted to normalize effector and regulatory CD4 T cell function. If our hypothesis is correct, miRNA-based therapies may not only prevent CNS autoimmunity in susceptible individuals, but ameliorate autoreactive T cells in patients with MS.